Evolution Of Tumor Cells Research Articles

Abstract 2095: AsiDNATM, a targeted therapy with no acquired resistance

Abstract The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity place the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we demonstrate that AsiDNA, the first antitumor drug with an agonist activity, can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to the strong alarm signal, generated by the AsiDNATM DNA repair inhibitors, did not promote resistance emergence. It induces a stable new state characterized by the down regulation of the targeted pathways that persists for months after treatments. This property is due to the original mechanism of action of AsiDNATM, which acts by over-activating a “false” signaling of DNA damage through DNA-PK and PARP enzymes, and is not observed with other DNA repair inhibitors such as the PARP inhibitors olaparib and talazoparib. Transcriptomic analysis of AsiDNATM treated independent populations derived from lung and breast cancers revealed the common evolution of independent AsiDNA-treated samples, resulting in a general down-regulation of enzymes involved in DNA repair and oncogenesis, and a specific down regulation of DNA-PK targets. Similar down regulation of DNA-PK targets was observed in xenografted tumor in animal models after three cycles of AsiDNA treatment. Chromatin analysis revealed a major change in AsiDNATM treated populations with an increased nucleosome stability that could be responsible for the epigenetic modification observed. Long term treatment with AsiDNATM induces a new “alarm down” state in the tumor cells that increase its efficacy. These results suggest that agonist drugs such as AsiDNATM could promote a state-dependent tumor cell evolution by lowering their ability to respond to damage signal as predicted by the ecological and evolutionary “smoke detector principle”. Citation Format: Wael Jdey, Maria Kozlac, Sergey Alekseev, Danielle Johnson, françoise Bono, Srividya bhaskara, frederic thomas, Marie Dutreix. AsiDNATM, a targeted therapy with no acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2095.

MODERN IDEAS ABOUT CLONAL EVOLUTION OF SARCOMAS

The progress in drug treatment of sarcomas is incredibly slow: primary, it is hindered by often developing resistance of tumors to standard chemotherapy with drugs in the maximum tolerated dosages. The clonal evolution of the tumor mass generates a high genetic heterogeneity of cancer cells, which underlies resistance to chemo- and radiotherapy. The consequence of such chemoresistance is local distribution and dissemination of the process, which, in turn, significantly reduces the quality of life of the patient and increases the probability of an adverse outcome of the disease. The purpose of this article is to highlight the modern understanding of the clonal evolution of tumor cells and the effect of this process on the clinical prospects of cancer patients.

Thresholds for extinction and proliferation in a stochastic tumour-immune model with pulsed comprehensive therapy

Periodical applications of immunotherapy and chemotherapy play significant roles in cancer treatment and studies have shown that the evolution of tumour cells is subject to random events. In order to capture the effects of such noise we developed a stochastic tumour-immune dynamical model with pulsed treatment to describe combinations of immunotherapy with chemotherapy. By using theorems of the impulsive stochastic dynamical equation, the tumour free solution and the global positive solution of the proposed system were investigated. We then show that the expectations of the solutions are bounded. Furthermore, threshold conditions for extinction, non-persistence in the mean, weak persistence and stochastic persistence of tumour cells are provided. The results reveal that comprehensive therapy or noise can dominate the evolution of tumours. Finally, biological implications are addressed and a conclusion is presented.

The Extinction and Persistence of Tumor Evolution Influenced by External Fluctuations and Periodic Treatment

This paper investigates the extinction and persistence of tumor evolution influenced by external fluctuations and periodic treatment. Firstly, a mathematical model describing the evolution of tumor cells with immunization under external fluctuations and periodic treatment is established based on stochastic differential equation. Then, making use of the methods of Ito’s formula, the sufficient conditions for extinction and persistence are derived by rigorous mathematical proofs. Finally, numerical simulations are applied to illustrate and verify the conclusions. The results of this work provide the theoretical basis for designing more effective and precise therapeutic strategies to eliminate cancer cells, especially for combining the immunotherapy and the traditional tools.

Abstract P3-01-09: Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient

Abstract Background: Copy number variation analysis has been shown as a valuable tool to infer clonal evolution of tumor cells. While bulk genomic analysis of FFPE tissue can provide information about the characteristics of a primary tumor or a metastatic nodule, these biopsies are rarely repeated due to their invasive nature. Liquid biopsies on the other hand, provide minimally invasive access to tumor derived cells and DNA and can therefore be used to monitor genomic changes of the tumor longitudinally and in real time. Here we evaluate the kinetics of liquid biopsy biomarkers and monitor genomic changes in single circulating tumor cells (CTCs) and cell-free DNA (cfDNA) from 17 longitudinal blood draws collected from a metastatic breast cancer patient over seven treatment regimes. FFPE tissue from the primary tumor and two metastatic sites in liver and bone are available for comparative analysis. Methods: We used the high definition – single cell assay (HD-SCA) platform to detect and enumerate CTCs of the blood of one metastatic breast cancer patient followed for 4 years. CTCs were characterized morphometrically and single CTCs of each draw were sequenced for copy number variation (CNV). cfDNA was extracted from plasma and tissue samples of the primary breast, bone and liver metastasis were micro-dissected. Extracted DNA from tissue samples and plasma underwent CNV analysis and were compared with CNV data of CTCs. Results: CTCs were identified in 14/17 draws and varied from 0 to >3000 cells/ml, spiking at two different times concordant with clinical progression. Changes in cell free tumor DNA fraction (ctDNA) correlated strongly with CTC count. Comparative CNV analysis of CTCs, cfDNA and FFPE tissue confirmed the tumorous origin of both cells and cfDNA. Single cell CNV analysis found four genomically related sub-clones in the patient's blood, three of which were present at time of enrollment, while the fourth clone emerged 3.5 years later concordant with the decline in the patient's health. Genomic copy number losses included tumor suppressor genes such as ARID1A, FOXP1, ATM, BRCA2, RB1, CDH11 and CDH1. Genomic gains were limited but included the proto-oncogenes BCL9, ABL2 and MDM4. It is striking that the sub-clonal structure persisted nearly unchanged for 3.5 years throughout various hormone and cytotoxic treatments, indicating high genomic stability. Despite their genomic differences, cells from the different clones are indistinguishable by immunofluorescence or morphometric analysis. CNV profiles of cfDNA reflect the most abundant CTC clone at each time-point. Conclusion: Using the HD-SCA assay cellular (CTC), and soluble (cfDNA) fractions of liquid biopsies can be compared at the molecular level over a long time course, and verified by comparison with tissue biopsy. This work confirms that liquid biopsies can be used to access the molecular state of a patient's cancer in near real-time and provide insight into the response to treatment years after the original characterization of the primary tissue. Here we show in a single patient example the power of single CTC analysis and highlight how CNV analysis of liquid biopsies could serve as minimally invasive tool to monitor tumor evolution longitudinally. Citation Format: Welter L, Xu L, McKinley D, Dago AE, Restrepo-Vassalli S, Rodriguez Lee M, Kolatkar A, Nieva J, Hicks J, Kuhn P. Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-09.

Abstract P2-03-11: Genetic background determines the algorithm of effectiveness of targeted drugs of RAS and PI3K pathways in TNBC: Testing a combination of MEK 1/2 inhibitor with mTOR kinase inhibitor or AKT inhibitor

Abstract Introduction: Most TNBC patients with single-agent targeted therapy in multiple clinical trials develop resistance, leading to disease progression, posing a major challenge in clinical management of mBRCA. In TNBC patients there remains a lack of obvious predictive biomarkers to guide targeted therapy. Although signaling mechanisms of resistance may be either intrinsic or acquired, the role of driver pathways are known be associated with oncogenic evolution of tumor cells to a resistant state. As the PI3K-AKT-mTOR1/2 (PAM) pathway is activated in TNBC (due to alterations in EGFR, 50%; PTEN, 40%; and PIK3CA, 5-10%), the PAM pathway is a candidate for potential molecular targeting of anti-cancer therapeutics. Despite the involvement of PI3K pathway activation in TNBC tumors, single agent PI3K inhibitors show modest clinical activity. Literature references suggest extensive cross-talk exists between the (PAM) and the RAS-RAF-MEK-ERK (MAPK) pathways. Here we tested the efficacy of a combination of MEK 1/2i (AZD6244, GDC0973) with mTOR kinasei (AZD2014, TAK228) or AKTi (AKT5363) in TNBC models. Methods: TNBC cell lines with alterations in KRAS/RAF (MDA-MB231), BRCA-deficient PTEN-loss (SUM149), PTEN-loss (MDA-MB468) and PIK3CA mutation (BT20) were used. Proliferation, apoptosis, cell cycle and 3D clonogenic growth were tested following drug treatment alone or in combination. Signaling events in the respective pathways following drug treatment were interrogated by WB. Results: MEKi led to a brief proliferation inhibition in MDA-MB231 cells. A combination of MEKi and mTORi was additive in decreasing short term proliferation and 10 day 3D growth. AKTi alone had limited effect on 3D growth but in combination with MEKi was profoundly inhibitory. A limited effect of MEKi was observed in BT20 cells; blocking 2D and 3D growth, while a profound effect of MEKi was observed in combination with mTORi (to a lesser extent with AKTi). In MDA-MB468 cells, MEKi did not have an initial appreciable effect (proliferation and G1 arrest), though 3D growth was significantly reduced at 10 days following drug alone or in combination with AKTi. SUM149 cells had G1 arrest with no appreciable change in 3D growth following MEKi. In contrast, single agent mTORi and AKTi reduced 3D growth. MEKi and mTORi doublet slowed proliferation, increased apoptosis and disrupted 3D growth. No single agent treatment with AKTi or mTORi disrupted colony formation however either agent in combination with MEKi blocked 3D growth. Single agents treatments were mostly cytostatic with no increase in apoptosis while a doublet of MEKi plus mTORi induced significant apoptosis.A combination of MEKi with PI3K pathway specific inhibitor significantly blocked phosphrylation of downstream effector molecules. Upregulation of ERK signals following PAM pathway inhibitors were abrogated by prior addition of MEKi. Summary: Combined blockade of the PAM and MAPK pathways (AKTi or mTORi) plus MEK1/2 inhibitor were more effective in attenuating molecular signals. This combination showed enhanced efficacy in TNBC cell models with specific PAM and or MAPK pathway alterations. Citation Format: Carlson JH, De P, Dey N, Leyland-Jones B. Genetic background determines the algorithm of effectiveness of targeted drugs of RAS and PI3K pathways in TNBC: Testing a combination of MEK 1/2 inhibitor with mTOR kinase inhibitor or AKT inhibitor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-11.

Pre-existing Functional Heterogeneity of Tumorigenic Compartment as the Origin of Chemoresistance in Pancreatic Tumors

Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that invitro cultures and invivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.

Hiding in Plain Sight: Epigenetic Plasticity in Drug-Induced Tumor Evolution.

Cancer is a heterogeneous disease with key differences at the cellular and molecular levels. Acquisition of these differences during the course of tumor development manifests into functional and phenotypic heterogeneity leading to tumor diversity, also referred to as intra-tumor heterogeneity (ITH). Within a tumor, there are subpopulations of cells capable of tumor initiation and maintenance. These cells often exhibit resistance to standard-of-care anti-cancer drugs. However, the role of various subpopulations (clones) in drug resistance remains to be investigated. Moreover, the jury is still out about whether drug resistance is a result of clonal selection of preexisting cells, or the cells acquire resistance by dynamic re-wiring of their epigenome. Therefore, we investigated the drug-induced tumor evolution in patient-derived primary cells of head and neck squamous cell carcinoma. Our data demonstrated the role of a preexisting poised epigenetic state in drug-induced adaptive evolution of tumor cells. Importantly, the combination of chemotherapy and epigenetic inhibitors can prevent/delay drug-induced tumor evolution.

Competitive evolution of NSCLC tumor clones and the drug resistance mechanism of first-generation EGFR-TKIs in Chinese NSCLC patients

PurposeAlthough many studies have reported on the resistance mechanism of first-generation EGFR TKIs (1st EGFR TKIs) treatment, large-scale dynamic ctDNA mutation analysis based on liquid biopsy for non-small cell lung cancer (NSCLC) in the Chinese population is rare. Using in-depth integration and analysis of ctDNA genomic mutation data and clinical data at multiple time points during the treatment of 53 NSCLC patients, we described the resistance mechanisms of 1st EGFR TKIs treatment more comprehensively and dynamically. The resulting profile of the polyclonal competitive evolution of the tumor provides some new insights into the precise treatment of NSCLC. Experimental designA prospective study was conducted in patients with advanced NSCLC with acquired resistance to erlotinib, gefitinib or icotinib. By liquid biopsy, we detected mutations in 124 tumor-associated genes in the context of drug resistance. These 124 genes covered all tumor therapeutic targets and related biological pathways. During the entire course of treatment, the interval between two liquid biopsies was two months. ResultsUnlike the common mutations tested in tissue samples, our data showed a higher coverage of tumor heterogeneity (32.65%), more complex patterns of resistance and some new resistance mutation sites, such as EGFR p.V769M and KRAS p.A11V. The major resistance-associated mutations detected were still EGFR p.T790M (45.28%), other point mutations in EGFR (33.9%), and KRAS and NRAS mutations (15.09%). These mutation ratios might be considered as a preliminary summary of the characteristics of Chinese patients. In addition, starting from the two baseline mutations of the EGFR gene (19del vs. L858R), we first described the detailed mutation profile of the EGFR gene. Although there was no significant difference in the number of patients with EGFR p.19del and EGFR p.L858R baseline mutations (24% vs. 16%, P = 0.15), patients from the EGFR p.19del baseline group were much more likely to develop EGFR p.T790M resistance mutations (62.1% vs. 19.3%, P = 0.007). Through careful integration of gene mutation information and clinical phenotype information, an interesting phenomenon was found. Although the variant allele fraction (VAF) of the EGFR p.T790M mutation was significantly linearly correlated with that of the EGFR drug-sensitive mutation (r = 0.68, P = 0.00025), neither VAF was associated with the tumor volume at the advanced stage. It was shown that other tumor clones might contribute more to the resistance to 1st EGFR TKIs treatment than tumor clones carrying the EGFR p.T790M mutation when resistance developed. By further analysis, we found that, in some patients, when the primary tumor clones detected were those carrying EGFR−/− mutations (both types the EGFR p.19del/p.L858R and EGFR p.T790M mutation types were missing), most of them showed a poor prognosis and ineffective late treatment, indicating that EGFR−/− played a more important role than EGFR p.T790M in the process of NSCLC drug resistance in these patients. From the perspective of the clonal evolution of NSCLC tumor cells, these phenomena could be explained by the competitive evolution between different tumor clones. In addition, two new mutations, KRAS p.A11V and EGFR p.V769M, emerged significantly during drug resistance in NSCLC patients and had shown obvious competitive clonal evolution characteristics. Combined with clear clinical drug resistance phenotypic information, we believed that these two new mutations might be related to new drug resistance mechanisms and deserve further study. We have also seen an interesting phenomenon. In some patients undergoing 1st EGFR TKIs treatment, the EGFR p.T790M mutation appeared, disappeared, and reappeared, and this spatial and temporal diversity of the EGFR p.T790M mutation was regulated by targeted drug and chemotherapy and was correlated with the individual tumor mutation profile. ConclusionsThe constitution and competitive evolution of the tumor clones have a decisive influence on treatment and can be regulated by targeted drugs and chemotherapy. Additionally, EGFR p.T790M spatial and temporal diversity during treatment warrants more attention, and this spatial and temporal diversity may be useful for the choice of treatment strategies for certain NSCLC patients. Through longitudinal cfDNA sample analysis, the resistance mechanism and dynamic clinical features of Chinese NSCLC patients are systematically established as reliable and meaningful to understand acquired resistance and make further personalized treatment decisions dynamically. Two new potential drug resistance-associated mutations in EGFR and KRAS have been found and are worthy of further study. Finally, our research shows that the evolutionary process of tumor cloning can be artificially regulated and intervened, possibly providing a new way to treat tumors.

Emergence of Intrahepatic Cholangiocarcinoma: How High-Throughput Technologies Expedite the Solutions for a Rare Cancer Type.

Intrahepatic cholangiocarcinoma (ICC) is the cancer of the intrahepatic bile ducts, and together with hepatocellular carcinoma (HCC), constitute the majority of primary liver cancers. ICC is a rare disorder as its overall incidence is < 1/100,000 in the United States and Europe. However, it shows much higher incidence in particular geographical regions, such as northeastern Thailand, where liver fluke infection is the most common risk factor of ICC. Since the early stages of ICC are often asymptomatic, the patients are usually diagnosed at advanced stages with no effective treatments available, leading to the high mortality rate. In addition, unclear genetic mechanisms, heterogeneous nature, and various etiologies complicate the development of new efficient treatments. Recently, a number of studies have employed high-throughput approaches, including next-generation sequencing and mass spectrometry, in order to understand ICC in different biological aspects. In general, the majority of recurrent genetic alterations identified in ICC are enriched in known tumor suppressor genes and oncogenes, such as mutations in TP53, KRAS, BAP1, ARID1A, IDH1, IDH2, and novel FGFR2 fusion genes. Yet, there are no major driver genes with immediate clinical solutions characterized. Interestingly, recent studies utilized multi-omics data to classify ICC into two main subgroups, one with immune response genes as the main driving factor, while another is enriched with driver mutations in the genes associated with epigenetic regulations, such as IDH1 and IDH2. The two subgroups also show different hypermethylation patterns in the promoter regions. Additionally, the immune response induced by host-pathogen interactions, i.e., liver fluke infection, may further stimulate tumor growth through alterations of the tumor microenvironment. For in-depth functional studies, although many ICC cell lines have been globally established, these homogeneous cell lines may not fully explain the highly heterogeneous genetic contents of this disorder. Therefore, the advent of patient-derived xenograft and 3D patient-derived organoids as new disease models together with the understanding of evolution and genetic alterations of tumor cells at the single-cell resolution will likely become the main focus to fill the current translational research gaps of ICC in the future.

Abstract 2851: The evolution of tumor cells under AsiDNA treatment results in “autosensitization”

Abstract Purpose: The Achilles' heel of all conventional and targeted anticancer treatments is intrinsic or acquired resistance. The era of precision medicine where tumors are selected for specific treatments based on their genetic alterations is revolutionary, but unfortunately prolonged responses are rarely observed due to rapid emergence of resistant clones. We recently developed a new concept of DNA repair inhibitor (Dbait) acting by activating enzymes involved in DNA damage signaling. We tested how such agonist activity would be prone to induce resistance to Dbait treatment. Experimental design: We performed repeated cycles of treatment with various targeted therapy agents (Imatinib, Olaparib, 6-thioguanine and the clinical form of Dbait, AsiDNATM). We analyzed the specific sensitivity of independent cultures after each treatment cycle. The study was performed in different tumor cell lines ((MDAMB231, MDAMB468, HCC1143, THP1, U937, KBM7) and in two non-malignant breast cell lines (MCF10A, MCF12A). Extensive transcriptome and genome studies were performed on MDAMB231 breast cancer cell lines. Results: Resistant clones appeared at a frequency of 1.45% for Olaparib, 0.62% for imatinib, and 1.66% for 6-thioguanine. Similar protocol did not select for resistance to AsiDNA. Unexpectendly, tumor cells became more sensitive to AsiDNA after each cycle of treatment. This behavior was specific of AsiDNA and was not observed with other treatments. The six tumor cell lines tested developed AsiDNA autosensitisation and no resistance. Non tumoral cells were not affected by repeated treatments. The acquired sensitivity of the treated tumor populations was conserved for months after end of treatment Transcriptional and genetic analysis of independently treated MDAMB-231 populations reveals that all evolved similarly. They display a few conserved genome modifications and a large deregulation of 1160 genes resulting in an overexpression of luminal associated genes and a decrease in mesenchymal associated genes. Conclusion: Our results indicate a phenomenon of “autosensitisation”, along treatment which has never been described for anticancer treatment and could prevent development of resistance during treatment. Citation Format: Maria Kozlac, Wael Jdey, Pierre-Marie Girard, Françoise Bono, Marie Dutreix. The evolution of tumor cells under AsiDNA treatment results in “autosensitization” [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2851.

PO-336 Identification of SOX2 and SFRP2 as opposing regulators of glioblastoma heterogeneous subtypes

IntroductionGlioblastomas display high levels of intra-tumour heterogeneity. It has recently been shown that individual glioblastomas may contain an intermixture of cells that display gene expression profiles representing the four gene expression based subtypes: proneural, neural, classical and mesenchymal. We have previously identified gene expression signatures that define two subtypes of glioblastoma cell cultures, where one represents a stem like, type A, and one a mesenchymal signature, type B, connected with a high or low capacity to grow as neurospheres and form intracranial tumours, respectively. Type A cultures are also defined by their expression of GFAP, and type B cultures by FN1. However, the relationship and regulation of these states is not well understood. In this study we aimed to identify genes that transition glioblastoma cells between a non-mesenchymal and mesenchymal cell state.Material and methodsTo identify transition genes we performed two overexpression screens of 27 candidate genes. Candidate genes, selected based on high differential expression between type A and type B cultures, were screened based on their capacity to alter FN1 or GFAP expression levels. Identified candidate genes where subsequently further tested for the capacity to alter gene expression, sphere forming capacity, anchorage independent growth, cell motility and cell growth.Results and discussionsWe identified SOX2 and SFRP2 as genes with opposite effects on GFAP and FN1 expression levels. SOX2 had the capacity to shift cell from a mesenchymal to a non-mesenchymal glioblastoma subtype profile. Global gene expression changes occurred where expression of progenitor related genes increased and mesenchymal related genes decreased. This was connected with increased tumour sphere formation, anchorage independent growth and reduced Matrigel invasion capacity. Conversely, SFRP2 increased FN1 expression, through an identified mechanism where it ablated SOX2 expression and thus reversed SOX2 induced phenotypes.ConclusionWe conclude that SOX2 and SFRP2 are opposing regulators of glioblastoma subtypes. Overall, these findings illustrate how the transition of glioblastoma cells from non-mesenchymal to a mesenchymal gene expression signature can be regulated during tumour cell evolution. Further understanding of the regulation of cell states in tumours with intratumoral heterogeneity and the interplay between cell populations is important for identifying targetable cancer driver pathways.

Abstract 1576: Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis

Abstract Tumor heterogeneity is prevalent in both treatment-naïve and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using High Definition Single Cell Analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management. Citation Format: Paymaneh D. Malihi, Michael Morikado, Lisa Welter, Anders Carlsson, Carmen Ruiz Velasco, Anand Kolatkar, Mariam Rodriguez-Lee, James Hicks, Peter Kuhn, Sandy T. Liu, Eric T. Miller, Radu M. Cadaneanu, Beatrice S. Knudsen, Michael S. Lewis, Isla P. Garraway. Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1576.

Abstract 1177: Clonal dissection of pancreatic tumors unmasks functional and genomic heterogeneous long-term self-renewing compartments at the origin of treatment resistance

Abstract Intrinsic and adaptive drug-resistance mechanisms allow human tumors to evade treatment through the demonstrated expansion of treatment-resistant clones. Thus, tumors are complex, dynamic ecosystems wherein populations of cells harboring both founder clones and unique, subclonal mutations coexist and progressively evolve. Modeling this functional heterogeneity of tumors can uncover critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long-term self-renewing cells that can be used to establish Clonal Replica Tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge across different animals over time, we uncovered that the long term self-renewing compartment of pancreatic cancer is represented by a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. Consistent with the stem cell hypothesis, although tumors respond to treatments and undergo a transient regression, their clonal complexity at the time of relapse is only partially compromised, implying that many tumorigenic cells survive the treatment and sustain tumor relapse. Moreover, our ability to track the same cell populations in different animals enabled us to demonstrate that the clonal composition of relapsed pancreatic tumors varied across the different drug treatment groups (gemcitabine, MEK1 inhibitor and dual PI3K/mTOR inhibitor), suggesting that the compartment of long-term self-renewing tumorigenic cells is highly functionally diverse in mediating drug resistance to different therapies. Notably, high-throughput isolation and deep characterization of unique clonal lineages isolated through CRTs demonstrated that individual self-renewing populations display a remarkable genetic and molecular heterogeneity that can account for the differential functional responses and adaptation to perturbations. So, our findings portend a model in which the genomic and functional heterogeneity within human tumors is maintained, propagated and recapitulated entirely by distinct pools of long-term self-renewing cells. This concept has important implications for the efficacy of pharmacological combinations, which has historically been ascribed to their synergistic effects to abrogate the emergence of resistance, may instead be linked to the ability of mechanistically unrelated drugs to delay relapse by targeting multiple populations of tumorigenic cells simultaneously. Citation Format: Sahil Seth, Chieh-Yuan Li, I-Lin Ho, Denise Corti, Sara Loponte, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Alessandro Carugo, Andrea Viale. Clonal dissection of pancreatic tumors unmasks functional and genomic heterogeneous long-term self-renewing compartments at the origin of treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1177.

Effect of cellular de-differentiation on the dynamics and evolution of tissue and tumor cells in mathematical models with feedback regulation

Tissues are maintained by adult stem cells that self-renew and also differentiate into functioning tissue cells. Homeostasis is achieved by a set of complex mechanisms that involve regulatory feedback loops. Similarly, tumors are believed to be maintained by a minority population of cancer stem cells, while the bulk of the tumor is made up of more differentiated cells, and there is indication that some of the feedback loops that operate in tissues continue to be functional in tumors. Mathematical models of such tissue hierarchies, including feedback loops, have been analyzed in a variety of different contexts. Apart from stem cells giving rise to differentiated cells, it has also been observed that more differentiated cells can de-differentiate into stem cells, both in healthy tissue and tumors, aspects of which have also been investigated mathematically. This paper analyses the effect of de-differentiation on the basic and evolutionary dynamics of cells in the context of tissue hierarchy models that include negative feedback regulation of the cell populations. The models predict that in the presence of de-differentiation, the fixation probability of a neutral mutant is lower than in its absence. Therefore, if de-differentiation occurs, a mutant with identical parameters compared to the wild-type cell population behaves like a disadvantageous mutant. Similarly, the process of de-differentiation is found to lower the fixation probability of an advantageous mutant. These results indicate that the presence of de-differentiation can lower the rates of tumor initiation and progression in the context of the models considered here.

Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis.

Tumor heterogeneity is prevalent in both treatment-naïve and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using high definition single cell analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management.

Kdm4c is Recruited to Mitotic Chromosomes and Is Relevant for Chromosomal Stability, Cell Migration and Invasion of Triple Negative Breast Cancer Cells.

Members of the jumonji-containing lysine demethylase protein family have been associated with cancer development, although their specific roles in the evolution of tumor cells remain unknown. This work examines the effects of lysine demethylase 4C (KDM4C) knockdown on the behavior of a triple-negative breast cancer cell line. KDM4C expression was knocked-down by siRNA and analyzed by Western blot and immunofluorescence. HCC38 cell proliferation was examined by MTT assay, while breast cancer cells’ migration and invasion were tested in Transwell format by chemotaxis. Immunofluorescence assays showed that KDM4C, which is a key protein for modulating histone demethylation and chromosome stability through the distribution of genetic information, is located at the chromosomes during mitosis. Proliferation assays demonstrated that KDM4C is important for cell survival, while Transwell migration and invasion assays indicated that this protein is relevant for cancer progression. These data indicate that KDM4C is relevant for breast cancer progression and highlight its importance as a potential therapeutic target.

Abstract A089: Exploiting clonal evolution and liquid biopsy to overcome resistance to anti-EGFR treatment in metastatic colorectal cancer: the CHRONOS trial

Abstract Background: The EGFR-targeted antibodies cetuximab and panitumumab (MAB-EGFRs) are used for the treatment of RAS-axis wild-type metastatic colorectal cancer (mCRC). Unfortunately, acquired resistance to MAB-EGFRs inevitably develops, dramatically curtailing their clinical use (Misale et al., Cancer Discov 2014). We found that mutations in KRAS, NRAS, and BRAF are the main culprit of this acquired resistance, and that high ctDNA levels of these mutated genes can be found in plasma of progressing patients. However, in preclinical models made resistant to cetuximab, acquired RAS-axis mutated clones decay upon cetuximab withdrawal, allowing tumor cells to regain sensitivity to further anti-EGFR treatment. Paralleling this behavior, RAS-axis WT patients who occasionally benefited from a rechallenge treatment with MAB-EGFRs exhibited corresponding pulsatile levels of mutant RAS ctDNA (Siravegna et al., Nat Med 2015). Collectively, these results suggest that the CRC genome adapts dynamically to intermittent MAB-EGFRs schedules, and provide a molecular rational for rechallenge therapies based on EGFR blockade. Methodology: CHRONOS is a liquid biopsy-driven trial to assess the efficacy of rechallenging with panitumumab (PAN) mCRC patients with ct-DNA-proven RAS-mediated acquired resistance. CHRONOS patients’ selection is based on a progressive enrichment strategy. In the SCREENING phase, multiple wild-type patients [MWTP, i.e., patients not harboring any drivers of resistance to MAB-EGFRs at tumor level (Bertotti et al., Nature 2015)] are identified upfront with a multiplex mCRC-specific tissue-genotyping panel (FUNNEL). In the MOLECULAR phase, MWTP responding to a 1st-line treatment inclusive of MAB-EGFR and developing RAS-mutated ctDNA clones at progression (PD), start 2nd-line chemotherapy and are blood monitored for RAS-related clones' decay. MWTP experiencing a &amp;gt;50% drop in ctDNA RAS levels at 2nd-line PD enter the TRIAL phase and are rechallenged with PAN. The main endpoint is objective response (OR), aiming to achieve a 30% OR rate. 129 MWTP responding and progressing to 1st-line MAB-EGFR-based therapy will be blood-monitored from first to second PD. We expect to find detectable (&amp;gt;3 MAF) ctDNA RAS mutated clones in 80% of cases (N 36), decaying &amp;gt;50% during 2nd-line "EGFR-therapy holiday" (60% of cases). These “ctDNA eligible” patients will receive PAN at 6 mg/kg Q2 weeks until PD or unacceptable toxicity. 27 MWTP will be recruited in TRIAL phase with 6 responses necessary to declare the study positive (β 0.15; α 0.05). Liquid biopsy will be used to correlate the decay kinetics of RAS-extended clone(s) during MAB-EGFR treatment holiday with response to PAN rechallenge, and to link the blood presence of RAS-extended clone(s) during PAN treatment with response and its duration. The tumor ctDNA plasma will be analysed by NGS before and after PAN rechallenge and potential associations identified between different mutated ctDNA clones (RAS or others) and response to PAN rechallenge. Conclusion: This is the 1st trial in mCRC exploiting liquid biopsy and the clonal evolution of tumor cell under treatment pressure, to extend the use of anti-EGFR therapy beyond the point of clinically established resistance in mCRC. (NCT03227926.) Funded by AIRC grant #9970. Citation Format: Salvatore Siena, Alberto Bardelli, Andrea Sartore-Bianchi, Sara Lonardi, Francesco Leone, Francesca Bergamo, Giuseppe Tonini, Filippo De Braud, Filippo Pietrantonio, Lorenza Rimassa, Armando Santoro, Giulia Siravegna, Benedetta Mussolin, Francesco Rua, Erica Francesca Bonazzina, Alessio Amatu, Patrizia Racca, Andrea Ardizzoni, Vittorina Zagonel, Silvia Marsoni. Exploiting clonal evolution and liquid biopsy to overcome resistance to anti-EGFR treatment in metastatic colorectal cancer: the CHRONOS trial [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A089.

Effect of aspirin on tumour cell colony formation and evolution.

Aspirin is known to reduce the risk of colorectal cancer (CRC) incidence, but the underlying mechanisms are not fully understood. In a previous study, we quantified the in vitro growth kinetics of different CRC tumour cell lines treated with varying doses of aspirin, measuring the rate of cell division and cell death. Here, we use these measured parameters to calculate the chances of successful clonal expansion and to determine the evolutionary potential of the tumour cell lines in the presence and absence of aspirin. The calculations indicate that aspirin increases the probability that a single tumour cell fails to clonally expand. Further, calculations suggest that aspirin increases the evolutionary potential of an expanding tumour cell colony. An aspirin-treated tumour cell population is predicted to result in the accumulation of more mutations (and is thus more virulent and more difficult to treat) than a cell population of the same size that grew without aspirin. This indicates a potential trade-off between delaying the onset of cancer and increasing its evolutionary potential through chemoprevention. Further work needs to investigate to what extent these findings apply to in vivo settings, and to what degree they contribute to the epidemiologically documented aspirin-mediated protection.

Blood vessel tortuosity selects against evolution of aggressive tumor cells in confined tissue environments: A modeling approach.

Cancer is a disease of cellular regulation, often initiated by genetic mutation within cells, and leading to a heterogeneous cell population within tissues. In the competition for nutrients and growth space within the tumors the phenotype of each cell determines its success. Selection in this process is imposed by both the microenvironment (neighboring cells, extracellular matrix, and diffusing substances), and the whole of the organism through for example the blood supply. In this view, the development of tumor cells is in close interaction with their increasingly changing environment: the more cells can change, the more their environment will change. Furthermore, instabilities are also introduced on the organism level: blood supply can be blocked by increased tissue pressure or the tortuosity of the tumor-neovascular vessels. This coupling between cell, microenvironment, and organism results in behavior that is hard to predict. Here we introduce a cell-based computational model to study the effect of blood flow obstruction on the micro-evolution of cells within a cancerous tissue. We demonstrate that stages of tumor development emerge naturally, without the need for sequential mutation of specific genes. Secondly, we show that instabilities in blood supply can impact the overall development of tumors and lead to the extinction of the dominant aggressive phenotype, showing a clear distinction between the fitness at the cell level and survival of the population. This provides new insights into potential side effects of recent tumor vasculature normalization approaches.